Incorporation of biologically active molecules into bioactive glasses

ABSTRACT

Carriers comprising silica-based glass providing for the controlled release of biologically active molecules, their methods of preparation, and methods of use are disclosed. The carriers are prepared using a sol-gel-derived process. Biologically active molecules are incorporated within the matrix of the glass during production.

This is a division of application Ser. No. 08/406,047, filed Mar. 17,1995, abandoned, which is a continuation-in-part of Ser. No. 08/281,055,filed Jul. 27, 1994, abandoned.

FIELD OF THE INVENTION

The present invention relates to the incorporation of biologicallyactive molecules into the matrix of glass, in particular bioactiveglass, using a sol-gel-derived process of production.

BACKGROUND OF THE INVENTION

Musculoskeletal injuries have a substantial impact on the health andquality of life of millions of Americans. Delayed healing of andnon-unions of fractures represent a continuous orthopaedic challenge.The conventional way of treating these problems is to use bone plates orscrews in combination with autologous bone grafting.

As a natural composite material, autogenous bone graft has been shown tohave both osteoconductive and osteoinductive properties. In addition, itis a sterile, non-immunogenic and non-toxic material, which has theability to be fully incorporated into the fracture site. Notwithstandingthe long duration for their activity to develop, autogenous bone graftsare the gold standard by which synthetic composites are compared. Giventhat there is also a limited supply and harvest site morbidity ofautogenous bone graft material, there is significant motivation todevelop synthetic composites. To date, no synthetic bone graftsubstitutes have fully achieved the properties of autogenous bone graft.

Enhancing the rate and probability of fracture healing and the promotionof bone formation and healing of delayed and non-union fractures are ofgreat clinical significance. (NIH/AAOS sponsored workshop. BoneFormation and Bone Regeneration. Tampa, Fla.: American Academy ofOrthopaedic Surgeons, 1993). The large population of patients withdelayed unions and non-unions of bone, the large direct medical costs,and the societal costs related to their long term disability, highlightthe need for effective and improved methods of treatment.

Advances in materials science and the identification of osteogenic andosteoinductive growth factors have invited the investigation of neweralternatives for autogenous bone grafting. osteogenesis, which is theprocess of bone formation, involves both osteoconduction andosteoinduction. Osteoconduction is the process in which differentiatedbone-forming cells produce a bone matrix upon an existing substrate.Materials that promote this process are considered osteoconductive.Osteoinduction is the process by which undifferentiated mesenchymalprecursor cells are transformed into differentiated bone forming cells.Factors or materials that promote this process are considered to beosteoinductive.

Growth factors delivered by biologically active controlled releasecarriers have the potential for improved fracture healing and lowermorbidity, thereby resulting in improved patient care and a decrease inthe overall costs associated with fracture care. Similarly, the deliveryof antibiotics by such carriers, either alone or in addition to growthfactors, will help reduce the incidence of infections, which can furthercontribute to delays in healing. In fractures involving, for example,the spine, the incorporation of anti-inflammatory agents and analgesicswill help control inflammation, which can also delay the healingprocess, and contribute to patient comfort during the healing process.Additionally, the controlled release of such materials regardless of thebioactivity of the carrier would represent a distinct advantage overcurrent delivery methods and assist fixation of implants.

The ideal synthetic graft would be a scaffolding material that wouldstimulate bone tissue to grow in place of the scaffold as it degrades.(Damien et al., J. Applied Biomater. (1991) 2:187-20.) Syntheticmaterials intended as bone graft substitutes should have mechanical andother properties similar to those of bone, and should be biocompatiblewith the surrounding tissues. In order to provide a union across thefracture site they must serve not only as scaffolding materials butalso, similarly to native bone, have a stimulatory effect on bone tissueregeneration.

The currently used synthetic bone graft materials are consideredosteoconductive in that they elicit the formation of the bone matrix attheir surfaces. Furthermore, they lead to a contiguous interface withbone or are replaced by bone tissue. Such properties suggest a chemicalinteraction between these bioactive materials and the bone environment.Cells existing in the bone matrix environment exhibit a beneficialresponse to these materials.

The materials studied most for use as synthetic grafts have been calciumphosphate ceramics and bioactive glasses. Calcium phosphate ceramics(CPCs) are very similar in composition to the mineral phase of bone.Bioactive glass are capable of forming a hydroxyapatite layer on theirsurface that mimics the mineral phase of bone.

The most commonly used calcium phosphate ceramics include:hydroxyapatite (HA), in either dense or porous forms, and β-tricalciumphosphate (β-TCP). Hydroxyapatite is of limited effectiveness as agrafting material. When HA particulate material in porous and dense formwas evaluated as a grafting material in the alveolar ridge it was foundthat fibrous encapsulation formed in perosseous sites. Migration of theparticles was also found to be a problem. (Ducheyne P., J. Biomed.Mater. Res. (1987) 21(A2 Suppl):219.) Further, HA cannot be used as ascaffolding material since its rate of degradation is slow. (Cornell etal., Clin. Orthop. (1992) 297; and Radin et al., J. Biomed Mater. Res.(1993) 27:35-45.)

β-TCP, on the other hand, is a biodegradable material which isosteoconductive. However, its degradation rate has been found to be toofast to serve as an effective synthetic graft material in load-bearingsituations. (Damien et al., supra.) Thus, clinical evaluations andapplications of the HA and β-TCP materials, either dense or porous, havedemonstrated that both materials are limited by a lack of controlledrate of reactivity.

Bioactive glasses were first found to bond to living bone by Dr. LarryHench in the late 1960's. Since that time, more than ten groups aroundthe world have shown that glasses containing SiO₂, CaO, P₂ O, Na₂ O andother smaller amounts of oxides in various compositions bond to bone.(Ducheyne P., J. Biomed Mater. Res. (1987) 21(A2 Suppl):219; Hench, L.L., Ann. N.Y. Acad. Sci. (1988) 523:54; Andersson et al., J. BiomedMater. Res. (1991) 25:1019-1030; Andersson et al., J. Non-Cryst Solids(1991) 129:145-151; Boone et al., J. Biomed Mater. Res. (1989) 23(A2Suppl):183; Ducheyne et al., Clin. Orthop. Rel. Res. (1992) 76:102-114;Hench, L. L., J. Biomed Mater. Res. (1989) 23:685-703; Kokubo, T.,Biomaterials (1991) 12(2):155; and Rawlings, R. D., J. Mater. Sci.Letters (1992) 11:1340-1343.)

Bioactive glass-ceramics undergo surface corrosion reactions whenexposed to body fluids. These corrosion reactions form a silica-richsurface layer. This layer serves as a nucleation site for the depositionof calcium phosphate, which evolves into a thick hydroxyapatite layer.When in contact with bone forming cells, this layer will form the basisof the chemical bond between the glass and the bone matrix. (Ducheyne,supra; Hench (1988), supra; and Hench, (1989), supra.) Dr. Hench's 45S5bioactive glass has been the most extensively studied of the bioactiveglass-ceramics. Its composition by weight % is: 45% SiO₂, 24.5% CaO, 6%P₂ O₅ and 24.5% Na₂ O.

In U.S. Pat. No. 5,204,106 (incorporated herein by reference), 45S5glass in particulate form in a narrow size range was described as beingan effective bone graft substitute in the alveolar ridge model and asbeing well incorporated into the surrounding bone. The glass granuleswere described as causing the upregulation of osteoprogenitor cells toosteoblasts that actively lay down bone tissue. (Schepers et al., J.Oral Rehabil. (1991) 18:439-452.)

The following parameters are important for bone-bioactive syntheticgrafts: controlled resorption and reactivity, immersion inducedtransformation of the synthetic materials' surface into abiologically-equivalent hydroxyapatite-like mineral, relatively largesurface area, and porosity (to create a network for osteoblasticactivity). Bioactive glass can potentially be tailored to fit theseparameters. In addition, the following requirements are important for asuccessful delivery system for biologically active molecules: 1)controlled release of the molecules; 2) delivery of adequate amounts ofthe molecules; 3) rapid growth of bone tissue into the carrier; 4)biocompatibility, osteoconductivity, and osteoinductivity of the implantmaterial; and 5) resorption of the carrier once bone tissue hascompletely formed. (Lucas et al., J. Biomed Mater. Res. (1989) 23(A1Suppl):23.) No delivery system currently available meets all of thesecriteria. (Damien et al., supra; and Cornell and Lane, Clin. Orth. Rel.Res. (1992) 277:297-311.) Certainly, no delivery system results incontrolled delivery.

Attempts have been made to try to improve calcium phosphate ceramics byusing them as delivery vehicles for bone growth factors. To date, therehas been no success in incorporating growth factors into calciumphosphate ceramics in a way that will lead to a sustained release of theadded growth factor. Mostly, one achieves a "burst" release, which is arapid initial release of most of the material over a short period oftime. (Campbell et al., Trans. Orthop. Res. Soc., 40:775, 1994.)

Carriers made of β-TCP , or nonsoluble collagen, have been moderatelysuccessful when combined with bone morphogenetic protein in attaininggood acceleration of bone tissue healing. (Damen et al., J. Dental Res.(1989) 68:1355-1359) However, these systems have not been able toproduce a measurable, controlled release of growth factor for time spansapproaching those needed for bone tissue regeneration to span large bonefilling defects. In one study, large amounts of growth factors, i.e.greater than 50 milligrams, were required to fill defects greater thanthree (3) centimeters. (Johnson et al., Clin. Orthop. (1992)277:229237.)

In most of the systems studied with osteoconductive materials used ascarriers, the method of incorporation has been that of simple immersionof the material into a growth factor solution. The growth factor is thenadsorbed either onto the material surface or into the pore structure,but is then quickly released upon immersion in an aqueous solution in aburst effect. (Campbell et al., Trans. Orthop. Res. Soc., 40:775, 1994.)

Published application WO 92/07554 reports a material which can beimplanted in living tissue which has a biodegradation rate matching therate at which the tissue regenerates. It is reported that the materialmay include an active substance providing an extended therapeuticaleffect. The material includes a calcium phosphate, biodegradable oxideor polyoxide, and an active substance having amine groupings such asnetilmicin and/or gentamicin in sulphate form.

Published application WO 93/05823 reports a composition for stimulatingbone growth comprising at least one of FGF, TGF-β, IGF-II, PDGF, andtheir biologically active mutants and fragments, or bone extracts withcorresponding activity, or bone extracts with BMP activity, and asuitable application material.

United Kingdom Patent Application GB 2255907 A reports a delivery systemfor biologically active growth and morphogenetic factors comprising asolid adsorbent selected for its specific affinity for the factor andthe factor adsorbed thereon. In one embodiment, porous hydroxyapatite isspecified as the solid adsorbent.

U.S. Pat. No. 4,869,906 describes a resorbable porous tricalciumphosphate in which the pores are sealed with a filler mixture ofantibiotic and a filler.

U.S. Pat. Nos. 5,108,436 and 5,207,710 describe stress-bearingprostheses having a porous region in combination with an osteogenicfactor extract or a purified osteogenic inductive protein, optionally incombination with a TGF-β cofactor, in a pharmaceutically acceptablecarrier. The carrier is either a collagen composition or a ceramic. Theosteogenic factor extract is dispersed in the porous region. Otherprocedures for combining the stress-bearing member with theosteoconductive material including coating, saturation, applying vacuumforce to get the material into the pores, and air-drying orfreeze-drying the material onto the member. It is further described thatthe pharmaceutically acceptable carriers preferably include a matrixthat is capable of providing a structure for developing bone andcartilage. Some preferred pharmaceutically acceptable carriers listedinclude collagen, hydroxyapatite, tricalcium phosphate, and bioactiveglass. However, there is no description of a preparation containingbioactive glass as a pharmaceutically acceptable carrier.

U.S. Pat. No. 4,772,203 describes implants having a core and a matrix,with the matrix being at least partially resorbable. The resorbablematrix is one or both of bioactive and osteogenesis-inducing. Tricalciumphosphate, hydroxylapatite sic!, and bioactive glass are listed as suchmatrixes. It is further stated that if a resorbable matrix is employed,it is further possible to embed antibiotics in the latter.

U.S. Pat. No. 4,976,736 describes biomaterials useful for orthopedic anddental applications having a base portion of calcium carbonate and asurface layer of a synthetic phosphate such as hydroxyapatite. Oneadvantage asserted for hydroxyapatite is absorbency. It is furtherdescribed that antibiotics or growth factors can be introduced into thepore cavities of the implant or attached, respectively. Alternatively,the antibiotic or growth factor can be intermixed with a preferablybiodegradable polymer and injected or vacuum infiltrated into theporosity of the phosphate surfaced material.

Gombotz et al., J. App. Biomat., (1994) 5:141-150 describe theincorporation of transforming growth factor-β into a composite implantmade from poly(lactic-co-glycolic acid) and demineralized bone matrix.It is reported that the implants exhibited an inflammatory response withlittle mineralization or bone formation. Similar results were reportedin Meikle et al., Biomaterials, (1994) 15(7):513-521 with polyDL-lactide-co-glycolide discs having bone matrix extract incorporatedtherein.

U.S. Pat. No. 4,563,350 describes a composition suitable for inductivebone implants comprising a purified form of osteogenic factor inadmixture with a carrier having a percentage of non-fibrillar collagen.The factor is added to the collagen either in solution or gelatin formand stirred in dilute mineral acid for 1-2 hours at approximately 4° C.The material is then dialyzed and lyophilized.

Japanese Laid-Open Patent Publication No. 5253286 describes a bonerestoring material comprising Ca-containing glass powder and orcrystallized glass powder, an aqueous solution composed mainly ofphosphate, and a medical substance in release-controlled form. Themedical substance is described as being in particulate form and can becoated with materials capable of oppressing the releasing of thesubstance temporarily.

As can be seen from the foregoing, a carrier providing for thecontrolled release of biologically active molecules is needed. Suchmaterials which are additionally osteoconductive and/or osteoinductiveare also needed.

Bioactive glasses are osteoconductive but are usually formed bycombining the different oxides in a platinum crucible and melting themixture at a temperature of 1300°-14000° C. This is the melt-derived, orconventional, method of obtaining bioactive glasses. Such temperatures,however, would destroy the function of most biologically activemolecules during preparation.

Another method which can be used to synthesize bioactive glass is thatof sol-gel processing. Sol-gel synthesis of glasses is achieved bycombining a metal alkoxide precursor, such as tetraethylorthosilane(TEOS, Si(OC₂ H₅)₄ in the case of silica), with water and an acidcatalyst to produce a hydrolysis reaction with consequent polymerizationof the metal alkoxide species and production of a gel. This gel willconsist mostly of the metal oxide when dried and will attain theconsistency of glass.

Several investigators have reported the incorporation of proteins into asol-gel-type glass produced using silicon alkoxide precursors and waterwith a maintenance of function. Braun et al., J. of Non-CrystallineSolids, (1992) 147 and 148:739-743; Yamanaka et al., Chemistry ofMaterials, (1992) 4(3):495-497; Ellerby et al., Science, (1992)255:1113-1115; and Avnir et al., Encapsulation of Organic Molecules andEnzymes, Ch. 27, pp385-404, American Chemical Society (1992). Methodsfor synthesizing low temperature, low alcohol, low proton-concentrationsol-gels for enzyme incorporation are described. The incorporatedproteins maintained their functionality. However, the focus of suchprocedures was the immobilization of the protein within the sol-gel in amanner which retains the protein of interest within the gel. When thesol-gel material functions as a sensor, very small molecules, such asglucose, can pass through the pores for assay. The incorporation withinthe sol-gel provides for repeated use of the protein. Release of theprotein from the sol-gel was not desired and would actually be counterto maintenance of long-term activity.

In U.S. Pat. No. 5,074,916, alkali-free bioactive sol-gel compositionsbased on SiO₂, CaO, and P₂ O₅ are described. Compositions ranges are44-86, 4-46, and 3-15 weight percent, respectively. However, the processdescribed utilizes tempertures around 600°-800° C. Such a process istotally incompatible with the incorporation of biological molecules.

SUMMARY OF THE INVENTION

The present invention is directed to controlled-release carriers. In thecarriers according to the invention, biologically active molecules areincorporated within the matrix of a silica-based glass. We have foundthat a derivation of the sol-gel technique facilitates suchincorporation without negatively affecting subsequent activity of themolecules. In the case of pure silica glass, the release of thebiological molecules from the carrier is effected primarily by diffusionthrough the pore structure. In the instance the glass contains oxides inaddition to silicon, the release of biological molecules is effected bydiffusion and reaction when immersed in fluids such as, for example,body fluids.

The sol-gel derived technique allows extensive control of the glassultrastructure and, thus, further control over the timing and quantityof release of the biologically active molecules, such as drugs or growthfactors. Such carriers can be both osteoconductive and osteoinductivethrough the formation of a calcium phosphate surface layer (i.e.bioactive) and the release of protein factors that attract and stimulatemesenchymal cells to differentiate into bone forming cells on thecarrier surface, as well as increase the proliferation of osteoblasts inthe local area. The net effect can be the acceleration of bone tissueregeneration and reduction in the incidence of infection in the areaadjacent to the sol-gel/biologically active molecule carrier composite,making the same particularly attractive as implants. The bioactivecomposite materials have a synergistic effect in promoting boneformation and, as such, can serve as an acceptable substitute forautogenous bone graft material.

In one aspect, the present invention relates to a carrier for controlledrelease of biologically active molecules over time comprisingsilica-based glass having biologically active molecules incorporatedwithin the matrix of the glass.

In another aspect, the present invention relates to a method forpreparing silica-based glass having biologically active moleculesincorporated in the matrix comprising reacting a silicon metal alkoxidewith water and methanol in a molar ratio of from about 6:1 to about 20:1water/alkoxide, adjusting the pH to a value between 1 and 4.5, addingthe biologically active molecule, allowing the mixture to gel and age attemperatures from about 0° C. up to about 40° C., and then drying theaged gel at temperatures from about 15° C. to about 40° C.

In another aspect, the present invention relates to a method forpreparing silica-based glass having biologically active moleculesincorporated in the matrix comprising reacting a silicon metal alkoxideand other alkoxides with water and methanol, adjusting the pH to a valuebetween 1 and 4.5, adding the biologically active molecule, allowing themixture to gel and age at temperatures from about 0° C. up to about 40°C., and then drying the aged gel at temperatures from about 15° C. toabout 40° C.

In another aspect, the present invention relates to a method forpreparing pure silica glass having biological molecules incorporated inthe matrix comprising reacting a silicon metal alkoxide with water andmethanol in a molar ratio of about 10:1 water/alkoxide, amethanol/alkoxide molar ratio of about 1:1, adjusting the pH to a valuebetween 1.5 and 3, adding the biologically active molecule, allowing themixture to gel and age at temperatures from about 0° C. to about 40° C.,and then drying the aged gel at temperatures from about 15° C. to about40° C.

In another aspect, the present invention relates to a method fordelivering biological molecules to a bony defect comprising implanting amaterial comprising a controlled-release carrier of silica-based glasshaving biological molecules incorporated within the matrix of the glassin the bony defect.

In another aspect, the present invention relates to method fordelivering antibiotics in situ comprising contacting a sample withsilica-based glass having antibiotics incorporated within the matrix ofthe glass.

In another aspect, the present invention relates to a method forpreparing a controlled-release carrier comprising silica-based glasshaving a porous matrix and biologically active molecules incorporated insaid matrix comprising combining a silicon alkoxide and calcium alkoxideand mixing under an argon atmosphere for up to about 15 minutes withoutany water, alcohol, or acid, being added. The biologically activemolecules are then added to the mixture in acid and the mixture isallowed to gel and age at temperatures from about 0° C. to about 40° C.,and then dried at temperatures from about 15° C. to about 40° C. until aweight loss of from about 50 percent to about 80 percent is achieved.

In another aspect, the present invention relates to a pre-treatedcarrier comprising silica-based glass having biologically activemolecules incorporated within the matrix of the glass. The carrier hasbeen treated by immersion in a solution containing ions typical forinterstitial fluid for a period of up to about seven days prior to use.

In another aspect, the present invention relates to an improved implantfor filling a bony defect. The improved implant comprises a coating of asilica-based glass having biologically active molecules incorporatedwithin the matrix of the glass.

In another aspect, the present invention relates to a composition forvarying release rates of biologically active molecules comprisinggranules of carriers for controlled release of biologically activemolecules over time comprising silica-based glass having biologicallyactive molecules incorporated within the matrix of the glass. To effectthe varying release rate, granules of different sizes in the range fromabout 500 μmm to about 5 μm are included.

In another aspect, the present invention relates to a compositioncomprising different populations of granules of carriers for controlledrelease of different biologically active molecules over time. Thecomposition comprises silica-based glass having biologically activemolecules incorporated within the matrix of the glass, each populationhaving a different biologically active molecule incorporated therein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a scanning electron micrograph of silica-based glassimmersed in simulated physiological solution.

FIG. 2 depicts energy dispersive x-ray analysis of a nodule detected onsilica-based glass immersed in simulated physiological solution.

FIG. 3 depicts the release of vancomycin, over time, from granules anddiscs of pure silica glass immersed in a simulated physiologicalsolution.

FIG. 4 depicts the effect of concentration on vancomycin release versustime.

FIG. 5 depicts a comparison of the zones of bacteria inhibition ofvancomycin dissolved in simulated physiological solution and vancomycinreleased from pure silica glass.

FIG. 6 depicts the zone of bacteria inhibition size versus immersiontime and concentration of vancomycin released from pure silica glass.

FIGS. 7a and b depict the relationship between trypsin inhibitorconcentration and release through 7 weeks and 4 weeks, respectively.

FIG. 8 depicts the effect of incorporated content, 0.5 vs. 1.0 μg, onthe cumulative release of active TGF-β1 from granules dried to a 50%weight loss.

FIG. 9 depicts the effect of the degree of drying, 50 vs. 70% weightloss, from granules loaded with 1.0 μg TGF-β1.

FIG. 10 depicts the effect of SA/V, granules vs. disks, loaded with 1 μgTGF-β1 and dried to 50% weight loss.

FIG. 11 depicts release of active TGF-μ1, per time period andcumulative, from disks loaded with 0.5μg and dried to 57% weight loss(n=3).

FIG. 12 depicts an absorption isotherm of silica-based glass containingother oxides.

FIG. 13 depicts FTIR spectra of silica-based glass containing otheroxides before (lower spectrum) and after (upper spectrum) immersion inSPS.

FIG. 14 depicts the release of trypsin inhibitor from sol-gelscontaining Ca and P.

FIG. 15 is an FTIR spectrum of a Ca-P sol-gel containing trypsininhibitor before and after immersion in a tris buffered electrolytesolution.

DETAILED DESCRIPTION

Utilizing the method according to the present invention, proteins andother biologically active molecules can be incorporated intosilica-based glass carriers in a way that leads to sustained release ofthe added molecules and does not destroy their function. Such acontrolled release delivery system can be used in implant materials, forexample, to fill in bony defects, including defects larger than threecentimeters without requiring an excessive quantity of growth factors.Such a controlled release delivery system also finds use in otherapplications with site-specific targeting needs such as, for example,chemotherapy. The carriers can be synthesized under sterile conditionsor can be sterilized subsequently using conventional sterilizationmethods.

Controlled-release carriers according to the invention comprisingantibiotics can be used in tissue culture for preventing contamination,particular that which develops upon consumption of antibiotic added withmedium, by contacting the carrier with the culture through, for example,immersion.

Controlled release carriers according to the invention comprising growthfactors, in particular bone growth factors, can be used to test theeffect of the continual, controlled release of different factors on bonecells in vitro. It is also contemplated that such carriers can be usedfor the development of immortal bone cell lines in vitro.

Sol-gel derived processing can be done at low temperatures--i.e.approximately 40° C. or below--and low pH. Both of these conditions canbe important for maintaining the functionality of biologically activemolecules incorporated into the sol-gel matrix.

The advantages of sol-gel derived processing include the following: 1) asol, which is a suspension of colloidal size particles, is in liquidform before it gels; 2) the whole reaction can be done at roomtemperature; and 3) the microporosity of sol-gel glasses can becontrolled by, for example, varying water content, timing of protonaddition, proton concentration, aging time, and drying time. The poresizes achievable with sol-gel processing in general are in the nanometerrange. During the liquid phase of the reaction, proteins and otherbiologically active molecules can be added to the liquid sol before itgels. These molecules then become encased in the solid matrix. Becauseof the controllable microporosity, a subsequent controlled release ofmolecule is achieved.

As used herein, "controlled-release carrier" refers to carriers forbiologically active molecules, as defined below, which provide for therelease of the biologically active molecules over time when immersed insolutions containing, for example, ions typical for interstitial fluid.An example of such a solution is simulated physiologic solution (SPS),used in some of the examples below. SPS is made by dissolving reagentgrade NaCl, KCl, NaHCO₃, K₂ HPO₄, CaCl₂, MgCl₂, and MgSO₄ in a 0.05MTris hydroxymethly!aminomethane hydrochloride (tris) buffered solution(pH 7.3 at 37° C.) resulting in ionic concentrations similar to plasma:Na⁺ =142 mM, K⁺ 5 mM Ca⁺² =2.5 mM, Mg⁺² =1.5 mM, HCO₃ =27 mM, HPO₄ ⁻² =1mM, and 0.5 mM SO₄ ⁻². Another example is tissue culture medium.

As used herein, "bioactive" refers to a bone bioactive material having acalcium phosphate rich layer present, or which develops duringappropriate in vitro or in vivo conditions. As observed by Pereira etal., J. of Biomed. Mat. Res., (1994) 28:693-698 (incorporated herein byreference), pure silica gel having a porous hydrated layer is able toinduce a carbonated hydroxyapatite layer when soaked in a simulated bodyfluid containing calcium and phosphate ions. Pure silica hydrogelsproduced using TEOS and drying temperatures of around 400° C. wereimmersed in simulated body fluids having different magnesium, calcium,and phosphate ions. It was reported that apatite nucleation inductionperiods were decreased with the addition of small amounts of calcium andphosphate ions to the fluids, as well as increase in pH. Li et al., J.Appl. Biomater., (1993) 4:221-229 and Li et al., J. Amer. Ceram. Soc.,(1993) 75:2094-2097 (both incorporated herein by reference).

As used herein, "silica-based" refers to the inclusion of a siliconoxide in the composition of the glass. Other oxides may also be present.

As used herein, "biologically active molecules" are defined as thoseorganic molecules having an effect in a biological system, whether suchsystem is in vitro, in vivo, or in situ. Biologically active moleculesinclude, but are not limited to, the following categories: growthfactors, preferably bone growth factors, cytokines, antibiotics,anti-inflammatory agents, analgesics, and other drugs. The term "type"as used hereinafter in reference to biologically active molecules refersto biologically active molecules of the previously listed categories, aswell as specific compounds, i.e. vancomycin, TGF-β, etc. These specificcompounds can be in the same or different categories.

The term "matrix" includes the solid framework of the bioactive glassstructure itself, as well as the pores. The phrase "incorporated withinsaid matrix" denotes that the molecules are incorporated throughout theglass network.

The term "bony defect" refers to regions necessitating repair including,but not limited to, fractures, areas of wear and tear, holes resultingfrom removal of screws and pins, replacements, periodontal applications,and deterioration of bone due to old age or disease.

The term "implant" refers to a material for filling bony defects asdescribed above. The implant preferably comprises a silica-based glassfurther comprising calcium. The implant can be in the form of granules,discs, blocks, or monoliths, and can comprise the controlled releasecarrier or simply be coated with the carrier. The implant can alsocomprise porous materials for use in bone surgery such as poroushydroxyapatite or, as described in WO 94/04657, porous bioactive glass.The term also includes prosthetic devices which, according to theinvention, can have a coating, or partial covering, of glass orbioactive glass having biologically active molecules incorporated withinthe matrix. Examples of such prosthetic devices include, but are notlimited to, hip and joint prostheses.

The implant can comprise a "cocktail" providing for a combination ofmaterials and/or release rates. The cocktail can include a population ofgranules of different sizes, all containing the same type ofbiologically active molecules. Alternatively, granules containingdifferent types of biologically active molecules can be combined. Thegranules in such a cocktail can be the same size or different sizes,thereby providing for the release of different molecules at differentrates. For example, a cocktail including antibiotics, anti-inflammatoryagents, and growth factors can be prepared.

It is also contemplated that two or more types of biologically activemolecules can be contained in each implant material as defined herein.This can be effected by simultaneous addition of the molecules into thesolution. Alternatively, implants containing one or more biologicallyactive molecules can be prepared and then these implants can,themselves, be coated with, or incorporated within, a solutioncontaining one or more different types of biologically active molecules,and/or at different concentrations.

The term "antibiotic" includes bactericidal, fungicidal, andinfection-preventing drugs which are substantially water-soluble suchas, for example, gentamicin, vancomycin, penicillin, and cephalosporins.

The term "growth factors" includes growth factors identified as havingosteogenic or osteoinductive properties. Included among the many factorsidentified with the control of bone formation are platelet derivedgrowth factors (PDGF), the transforming growth factors (TGF-β),insulin-like growth factors (IGFs), fibroblast growth factors (FGFs),and the bone morphogenetic proteins (BMPs). These growth factors arepresent at the site of fracture healing in vivo and are produced at thetime of injury through platelet lysis (PDGF and TGF-β) and by theresorption of bone matrix (TGF-β and BMPs). The individual factors willbe discussed in more detail below.

The term "contacting" includes, but is not limited to, contacting thecarrier with the sample for which release of the biologically activemolecules is targeted through, for example, immersion, implantation, andembedding.

The identification of osteogenic and osteoconductive growth factors hasspawned the search for new graft substances obtained through geneticengineering concepts. The controlled delivery of these recombinantmolecules, however, is important. Growth factors with known effect onbone tissue must be delivered at the site in sufficient doses tostimulate healing. Glasses synthesized following a room temperaturesol-gel-derived procedure are outstanding candidate materials for thecontrolled release of such osteoinductive molecules. The processing ofthe glass allows one to control the ultrastructure of the glass suchthat the timing and quantity of release are tailored to fit the specifictherapeutic needs. In addition, these glasses can be osteoconductive,thereby providing a substrate for bone tissue development.

The effects of the growth factors when exogenously applied to in vitroand in vivo experimental models of bone formation have demonstratedtheir biological properties. (Cornell et al., supra; and Mohan et al.,supra.) Consequently, any material which affords the sustained deliveryof such factors is beneficial. Although most of the previous studiesclearly demonstrate the osteogenic and osteoinductive effects of theseproteins, the precise biological properties of these growth factors withrespect to the degree of bone formation is greatly influenced by thefollowing: the environmental conditions of the experimental model, thetiming, method and dose of growth factor delivery, the hormonal milieauand the synergy between the various growth factors. Thus, the presentinvention provides a method to elucidate the effects of these growthfactors.

From a developmental point of view, the formation of bone occurs in aseries of discrete steps. Initially there is a proliferative phasefollowed by cellular differentiation and deposition of a collagenousmatrix which in itself influences subsequent expression of boneproteins. (NIH/AAOS sponsored workshop, supra.) Some workers viewcollagenous matrix synthesis as a series of temporal events in whichthere is an initial collagenous phase followed by a rise in alkalinephosphatase activity and the expression of osteonectin, bonesialoprotein and osteocalcin. Osteopontin expression and synthesis hasbeen further dissected temporally in terms of sulfation, phosphorylationand molecular size. Aside from the proteins listed above, other studieshave shown that at least two forms of chondroitin sulfate proteoglycanare also synthesized by the osteoblast. These parameters can all bemeasured by methods well known in the art. Some growth factors aredetailed below.

Insulin-like growth factor (IGF) I and II are made by bone cells as wellas by other tissues throughout the body. They are found in bone matrixand have presumably been secreted by bone cells. (Canalis et al.,Calcified Tissue Int. (1993) 53:S90-S93; and Canalis et al., J. BoneMiner. Res. (1993) 8:S237.) In vitro, IGFs have been shown to increasebone collagen and matrix synthesis, to increase osteoblast-precursorreplication and decrease bone collagen degradation. (Hock et al.,Endocrinology (1988) 122(1):254); and Mccarthy et al., Endocrinology(1989) 124(1):301.)

Growth hormone is thought to act through IGF in stimulating bone growth,but it has also been shown to have local effects on mesenchymal cellproliferation and differentiation. (Downes et al., J. Mater. Sci.:Mater.Med., (1991) 2:176-180; and Silbermann, M., Biomaterials, (1990)11:47-49.) Human growth hormone has two molecular weight species, one of20,000 and the dominant species of 22,000.

Platelet derived growth factor (PDGF), a polypeptide of approximately 30kD in molecular weight, exists as a dimer composed of two A subunits ortwo B subunits or as a heterodimer of an A and a B subunit, creatingthree separate forms of PDGF. These subunits are the products of twoseparate genes. While all three forms are found in bone matrix, onlyPDGF AA is made and secreted by bone cells in vitro. PDGF BB has beenfound to be the most active of the three forms. (Mohan et al, supra.)

PDGF has been shown to have bone resorbing activity in vitro; a numberof investigators have reported increased bone resorption in response toadministration of physiological doses of PDGF. (Tashjian et al.,Endrocrinology (1982) 111:118-124.) Additionally, PDGF has been shown toincrease osteoprogenitor cell replication.

Transforming growth factor-beta (TGF-β) is a family of molecules whichmay have bone promoting properties for fractures. TGF-β is a homodimericpeptide with a molecular weight of 25 kD. The most abundant sources ofthis molecule are platelets and bone. This multifunctional peptide has abroad range of cellular activities, including control of theproliferation and expression of the differentiated phenotype of severalcell types specific to bone, among them mesenchymal precursor cells,chondrocytes, osteoblasts, and osteoclasts. (Beck et al., J Bone Miner.Res. (1991) 6(9):961; Joyce et al., Orthop Clin. North Am. (1990)21(1):199; and Joyce et al., J. Cell Biol. (1990) 110(6):2195.) Althoughit exists in several distinct forms, two of these, TGF-β1 and 2, havebeen isolated from bone in approximately a 4:1 ratio. In vivo studiesbased on both immunohistochemical staining and in situ hybridizationhave demonstrated the synthesis of TGF-β by both chondrocytes andosteoblasts and the accumulation of TGF-β in models of endochondralossification. (Joyce et al., Orthop Clin. North Am. (1990) 21(1):199;and Joyce et al., J. Cell Biol. (1990) 110(6):2195.) In a study in whichTGF-β 1 or 2 was introduced by daily injection into the subperiostealregion of newborn rat femurs, (Joyce et al., J. Cell Biol. (1990)110(6):2195) demonstrated that mesenchymal precursor cells in theperiosteum were stimulated by TGF-β to proliferate and differentiate inmuch the same manner as that which is observed in embryological boneformation and early fracture healing. After the cessation of injections,endochondral ossification also occurred, resulting in the replacement ofcartilage with bone.

The implantation of a bone morphogenetic protein (BMP) solution leads toa series of developmental processes including chemotaxis, proliferation,and differentiation, which result in the transient formation ofcartilage and its replacement by living bone tissue complete withhematopoietic marrow. (Urist, M. R., Science (1965) 150:893-899.)Several newly discovered factors, BMP-1 through 7, and osteoinductivefactor (OIF) have been implicated in the BMP process. BMP-2 through 7are all members of the TGF-β superfamily of molecules and are closelyrelated to two factors Vg1 and DPP which are involved in a variety ofdevelopmental processes during embryogenesis. Both BMP-2A and BMP-7 havebeen expressed as recombinant proteins both of which have been shown toclearly induce the entire cartilage and bone formation process seen withbone-derived BMP solutions. (Wozney, J. M., Prog. Growth Factor Res.(1989) 1(4):267.) At the present time, two BMPS: BMP-2A (Gerhart et al.,Clin. Orthop. (1993) 317; Wozney et al., Science (1988) 242(4885):1528;and Yasko et al., J. Bone Joint Surg. <Am> (Aug. 1992) 74(7):1111 and J.Bone Joint. Surg. <Am> (1992) 74(5):659) and BMP-7 (Sampath et al., J.Biol. Chem. (1992) 267(28):20352) (also known as OP-1) have beendemonstrated to increase bone formation at extraosseous sites, and toenhance fracture healing. (Gerhart et al., Clin. Orthop. (1993) 317.)Purified BMP has been utilized in femoral and tibial non-unions inuncontrolled clinical trials. (Johnson et al., Clin. Orthop. (1988)230:257-265; Johnson et al., Clin. Orthop. (1988) 236:249-257; andJohnson et al., Clin. Orthop. (1990) 234.)

Current state of knowledge suggests that the local growth factors mostlikely to increase fracture healing significantly are PDGF, TGF-β andBMP-2.

Maintenance of function of growth factors after incorporation within thesilica-based glasses can be tested using the aforementioned techniquesfor determining bone differentiation. The maintenance of function ofantibiotics can be ascertained using standard disc susceptibility testssuch as are described in Antibiotics in Laboratory Medicine, 3rdedition, V. Lorian, ed., chapter 2, Williams and Wilkins, Baltimore,Md., 1991 (incorporated herein by reference). Function of incorporatedanti-inflammatory agents and analgesics can be ascertained by, forexample, testing for inhibition of prostaglandin synthesis in cellculture.

Sol-gel-derived glass synthesis

Pure silica and calcium containing glasses have been synthesized withbiologically active molecules incorporated therein. Briefly, a siliconalkoxide precursor, preferably tetramethylorthosilane (TMOS), in puresolution is combined with deionized water and stirred by magnetic orultrasonic means. The water to TMOS molar ratio affects porosity andspecific surface area of the gels, which, in turn, affect bioactivity.As both increase, so can bioactivity. To increase both, water isprovided in amounts exceeding stoichiometric, or in an H₂ O/TMOS molarratio ranging from about 6:1 to about 20:1. In a preferred embodimentthe molar ratio of H₂ O/TMOS is 10:1. Alcohol, preferably methanol, canbe added at an alcohol/TMOS molar ratio of from about 0:1 to about 1:1.Acetic acid (0.1N) or HCl (0.1N) can be used as a catalyst for thehydrolysis reaction, and is added to maintain the desired pH, asdisclosed below.

Calcium methoxyethoxide (20% solution in methoxyethanol, Gelest Inc.,Tullytown, Pa.) can be used as a calcium alkoxide source. Calciummethoxyethoxide (CME) is added in an amount sufficient to result in afinal percentage of up to about 40% by weight calcium oxide upon dryingof the gel. Triethyl phosphate can be used as a phosphorous pentoxidesource. Triethyl phosphate (TEP) can be added to achieve a finalconcentration of phosphorous pentoxide, P₂ O₅, up to about 10% by weightupon drying. Weight percentages throughout are calculated based upon thereactions going to completion and complete drying. The water, TMOS, andacid are mixed using sonication in an ice bath, or magnetic stirring, ora combination of both. When a calcium alkoxide is present, the TMOS,calcium alkoxide and additional alkoxides, if any, are preferably mixedunder non-aqueous conditions under an argon atmosphere using eithermagnetic stirring or sonication for up to about one hour.

Since the biologically active molecules to be incorporated retain theirbiological activities after treatment in moderate to highly acidicconditions, an amount of acid necessary to maintain acidity in a rangeof pH from about 1-4.5, preferably about 1.5-3, prior to, or during,incorporation of biologically active molecules is used.

The biologically active molecules to be incorporated are added atconcentrations resulting in final concentrations ranging from about0.0001 to about 10% by weight of the glass.

Glasses with compositions of silicon in the range of 60-100% (by weight)with the remainder as other oxides can be prepared. The liquid sol canbe cast into a polystyrene container. The sol is aged and allowed to gelin a sealed container. Aging can take from about one (1) day to aboutfour (4) weeks. Drying can be performed for a time of from about 1 toabout 14 days.

In order for sol-gel derived glass to be an effective carrier forbiologically active molecules, the process should be carried out at alow temperature (about 2°-40° C.) and, in the case of pure silica glass,the acidity of the sol should be between pH 1 and 4.5. Temperature, solpH, % calcium content, water to TMOS molar ratio and other factorsaffect the gelling time of the sol. However, when incorporatingbiologically active molecules, the gelling time of the sol should allowenough time in the liquid state to enable the addition of thebiologically active molecule solution for incorporation, cast, andhomogeneously mix the sol. Gelation occurs when enough cross-links haveformed such that the network spans the length of the container. Grossobservation reveals little or no movement of the cast material uponinversion.

A lower pH increases the gelling time. A higher calcium contentdecreases gelling time. A higher gelling time is desirable in order tosee more of the sol-gel reactions going to completion, thus ending witha final material with less porosity and smaller pore size. Less porosityalso means a more mechanically strong material with longer times ofprotein release. However, there are instances when greater porosity maybe desirable, for example, achieving a more rapid release of molecules,or a more rapid degradation of the carrier. Larger pore sizes facilitatethe release of larger molecules through diffusion.

A lower temperature also increases gelling times. To achieve lowertemperatures, the reaction is then carried out in an ice-cooled waterbath. A higher water content will also decrease gelling time for mostmetal alkoxides, although the porosity may stay high due to increasedwater evaporation from the material. Conditions are selected such thatgelation optimally occurs within a period ranging from at least about 30minutes to about 48 hours for incorporating biologically activemolecules. Gelation can be performed at temperatures ranging from about0° C. to about 40° C.

Aging of the sol-gel occurs after casting and is performed by keepingthe casting container sealed. Sol-gel reactions continue unimpededduring this period. Aging can be performed at temperatures ranging from0° C. to about 40° C. Longer aging times (of up to 1 month) result in amore mechanically strong material, which undergoes less cracking thanmaterials that have been aged for lesser time periods. Aging at a lowertemperature, such as 4° C., also extends the gelling time.

Drying temperature and time can also affect the final materialcharacteristics. A fast rate of drying can produce cracks in the finalmaterial. The final material loses about 50-80% of its weight betweencasting and final drying due to evaporation of water, and alcoholsproduced as by-products of the reaction. Drying is performed attemperatures ranging from about 15° C. to about 40° C. by unsealing thecasting container, and can be performed at atmospheric pressure, orpressures lower than atmospheric.

As is evident from FIGS. 3, 7, 8, and 4, the release kinetics of thebiologically active molecules in the early stages of immersion, i.e.from about one day to seven days, is higher than those in the laterstages. At about seven days after immersion, a major change in the slopeof the curves is observed, representing a major change in rate ofrelease. The early higher release is not a "burst" effect as previouslyreported by several authors (cited above). This higher early release isadvantageous when a dual treatment regimen is imposed--an acutetreatment at a high dose, followed by a "chronic" lower dose. In caseswhen a steady state release is desired right from the onset of themedical treatment, i.e., a release without major changes in rate, thesol-gel carriers can be treated by immersion at the time of productionsuch that the intitial higher release phase has taken place beforeactual use in the patient.

EXAMPLE 1 Synthesis of Sol-gel/Vancomycin Composite

A sol-gel derived silica-based matrix-vancomycin composite wassynthesized employing a room-temperature, low acidity, low alcoholconcentration procedure. Vancomycin was selected as the drug to bereleased due to its proven efficacy against gram positive cocci,especially staphylococci, which is a major cause of osteomyelitis.Vancomycin is a water soluble (up to 100 mg/ml) tricyclic glyceropeptideof approximately 3,300 molecular weight.

The material was prepared as follows: 19.6 ml tetramethylorthosilicate(TMOS, Aldrich, St. Louis, Mo., U.S.A.), 14.2 ml water, 5.2 methanol and0.01 ml of 1N HCl was sonicated in a glass beaker in an ice bath for 30minutes. Then, 4 ml of the sol was cast into 23 mm diameter polystyrenevials (Sarstedt, Princeton, N.J.) and 1 ml of 10 mg/ml vancomycin HCL(Lederle, Carolina, Puerto Rico) was added to the sols in the vials andthe samples were mixed. The same amount of water, i.e. 1 ml, was addedto control samples. The total H₂ O/TMOS ratio was 10:1. Themethanol/TMOS ratio was 1:1. The amount of incorporated vancomycin tosample weight was about 1%. The vials were sealed with airtight caps,gelled, aged, and dried at room temperature. Time to gelation variedfrom 15 to 25 hours. Addition of the vancomycin solutions did not changesignificantly the time to gelation.

After aging for 2 weeks in the sealed containers, the sols were exposedto air for drying. During drying, evaporation of liquid from the gelpore network resulted in weight loss and shrinking of the gels. Theweight loss continued up to 2 weeks. Drying was considered to becomplete when the weight loss reached 75-78%. The significant weightloss and shrinking did not produce visible cracks. The resultingproducts were transparent monoliths in a shape of 11 mm diameter and 8mm high cylinders weighing 1.1 gram. The density of the dried gelmaterial was equal to 1.5 g/cm³. Since 10 mg vancomycin was incorporatedinto each of the discs, the vancomycin content in the material was0.91%. There is no reason to expect that other water-soluble antibioticswill behave any differently.

EXAMPLE 2 Vancomycin Release Study

For the in vitro vancomycin elution study, a part of the monoliths wascrushed, ground, and sieved to obtain either small granules in a sizerange from about 500-700 μm, or large granules of about 5×5×2 mm. Therest of the monoliths were tested as discs.

The synthesized vancomycin composite was immersed into a simulatedphysiological solution (SPS) with ion content similar to that of plasmaas disclosed previously. To determine the effect of the sample surfacearea to volume (SA/V) ratio, the material used for the immersionexperiments was shaped as follows: small granules of 500-700 μm (SA/Vapproximately 10 mm), large granules 5×5×2 (SA/V=1.5 mm⁻¹), discs 11 mmdiameter×4 mm (SA/V=0.85 mm⁻¹), and half-cylinders 5.5 mm×4 mm (SA/V=1.2mm-1).

All the samples were immersed at the same vancomycin content insample/solution ratios equal to 1 mg vancomycin per 1 ml. The immersedsamples were incubated at 37° C. for time periods ranging from about 1hour to about 3 weeks. The solutions were totally exchanged at thefollowing time periods: 1 hour and 1, 3, 7, 14, and 21 days.

The released vancomycin concentrations were measured using an automatedFluorescent Polarizing Immunoassay system (TDxR system, AbbottDiagnostics, Irving Tex.). The results of the vancomycin release assayare presented in FIG. 3 and summarized in Table I below. In FIG. 3, opencircles represent the small granules. Open triangles represent the largegranules. Open squares represent the 11 mm diameter discs. Open invertedtriangles represent the 5.5 mm diameter discs.

                  TABLE I                                                         ______________________________________                                                                 %,                                                                 Release Time                                                                             Released/Incorporated                                Sample        (days)     Vancomycin                                           ______________________________________                                        Small Granules                                                                               6         100                                                  Large Granules                                                                              21         55                                                   Disc (SA/V = 1.2 mm.sup.-1)                                                                 21         48                                                   ______________________________________                                    

As indicated by the foregoing data, the vancomycin release rate wasaffected by the material shape, i.e. the material surface area to volumeratio. Specifically, the vancomycin release from the small granules wasvery rapid and most of the incorporated vancomycin was released duringthe first day of immersion. In contrast, the large granules (SA/V=1.5mm⁻¹) and discs (SA/V=1.1 or 0.8 mm⁻¹) showed a continuous vancomycinrelease, which started at one hour, gradually increased to a maximum,then slowly decreased, tailing-off up to 3 weeks later. The maximumvancomycin release was measured during the period of immersion betweenthree days and one week.

These findings indicate that the SA/V ratio can affect the release ofmaterials. Combination of the materials of varying shape, i.e. varyingSA/V ratio, can provide a controlled vancomycin release which startsupon immersion and continues for up to one month.

EXAMPLE 3 Effect of Vancomycin Concentration

The sol-gel derived silica based matrix-vancomycin composites withvarying vancomycin content were synthesized. The sols were prepared asdisclosed above in Example 1. Then, 1.2 ml of the sol were cast into 23mm diameter polystyrene vials. The cast sols were divided into twogroups and 0.3 ml of solutions with different vancomycin concentrationswere added to the cast sols of both groups in order to keep the same H₂O/TMOS molar ratio of 10:1. The amounts of the incorporated vancomycinwere 10 and 20 mg for groups 1 and 2, respectively. The percentage ofvancomycin to sample weight was equal to 2.8 and 5.5%, respectively. Thesols were gelled, aged, and dried to about 75% weight loss.

Ultrastructure parameters of the sols such as specific surface area(SSA), average pore size (PS), and pore volume (PV) of the dried solswere determined using the monolayer gas absorption technique (multipointB.E.T., Quantachrome). The measured values were as follows:

    ______________________________________                                                SSA, m.sup.2 /g                                                                             545                                                             PS, nm        1.8                                                             PV, cc/g      0.45                                                    ______________________________________                                    

The obtained sol-gel derived discs, 11 mm diameter×2 mm, with SA/V ratioequal to 1 mm⁻¹, were subjected to vancomycin release study as disclosedabove in Example 2. The discs were immersed into 5 ml SPS. Thevancomycin content in sample (total weight of vancomycin) to solutionvolume ratios (Wv/V) were 2 and 4 for groups 1 and 2, respectively. Theconcentrations of released vancomycin were measured as described abovein Example 2. The results of the study are presented in FIG. 4. In FIG.4, solid bars represent vancomycin at 10 mg incorporation. Hatched barsrepresent vancomycin at 20 mg incorporation.

The data show that the amount of released vancomycin increased with theamount of incorporated drug. Thus, the released amount appears to be afunction of the incorporated quantity (at conditions otherwise equal).However, the drug release profile over time appears to be similar fordifferent concentration. Specifically, the drug release started rightafter immersion, reached a maximum by 3 days, then gradually decreased.

EXAMPLE 4 In vitro Bacteria Inhibition Test

The SPS solutions with varying contents of vancomycin, released from thesol-gel derived silica-based matrix from the experiments described inExamples 2 and 3, were tested for susceptibility of Staphylococcusaureus bacteria to the released drug. The standard disc susceptibilitytest technique was applied (See Lorian, supra.). The sample SPSsolutions with vancomycin released during immersion were tested andcompared with standard solutions of vancomycin in SPS withconcentrations ranging from 100 to 10,000 μg/ml. Concentrations of thesample SPS solutions with vancomycin released during immersion weremeasured using the Fluorescent Polarizing Immunoassay describedpreviously. Single, twenty μl aliquots of each solution (either standardor sample) were deposited onto 1/2 inch filter paper discs (#740-E,Schleicher & Schnell, Keene, N.H.). The drug solution impregnated discswere then dried and stored in a desiccator at 4° C. A blood agar plateinoculated with Staphylococcus aureus (ATCC 25923) was obtained from theMicrobiology Laboratory, Hospital of the University of Pennsylvania. A1.5×10⁸ CFU/ml suspension of bacteria in 0.45% saline was created tomatch a McFarland Equivalence Turbidity Standard 0.5 (Remel, Lienexa,Kans.). Mueller-Hinton agar plates, 15×100 mm (Model 01-620, Remel,Lienexa, Kans.) were inoculated with 10 μl of the Staphylococcus aureussuspension by streaking with a sterile swab soaked in the suspensionover the entire agar surface to ensure an even distribution of inoculum(standard inoculation procedure). A vancomycin impregnated disc wasplaced in the center of each agar plate. The agar plates were thenincubated in a humidified air environment in a single-chamber,water-jacked incubator (Model 3159, Forma Scientific, Marrietta, Ohio)at 37° C., for 24 hours. Zones of bacteria inhibition were measuredusing a caliper with a precision of 0.1 mm. The data are presented inFIGS. 5 and 6.

The measured zone of inhibition sizes plotted against vancomycinconcentration in a logarithmic scale of vancomycin released fromsol-gel, as disclosed in Example 3, are presented in FIG. 5. In FIG. 5,open circles represent vancomycin dissolved in SPS. Closed circlesrepresent vancomycin released from the sol-gel carrier.

The discs, impregnated with 30 μg of vancomycin, either dissolved in SPSor released from the silica-based matrix, exhibited a zone size greaterthan 12 mm. According to the Zone Diameter Interpretive Standards(Lorian, supra, Tab. 2.1.), a zone of that size indicates that bacteriaare susceptible to the material, and the equivalent minimum inhibitoryconcentration breakpoint is less than 4 μg/ml. The concentration-zone ofinhibition relationship for the sample solutions of vancomycin releasedfrom the silica-based matrix showed a close fit to that of the standardsolutions.

FIG. 6 shows zone of bacteria inhibition sizes versus immersion time andconcentration of vancomycin incorporated into the sol-gel derived silicamatrix. Open bars represent vancomycin at 1 mg. Solid bars representvancomycin at 10 mg. Cross-hatched bars represent vancomycin at 20 mg.The data demonstrate that vancomycin released from the sol-gel matrixwas effective to inhibit the bacteria growth up to three (3) weeks (at20 mg concentration). The measured zone of inhibition sizes appear toincrease with concentration of incorporated vancomycin, reflectinglarger quantities of released vancomycin.

The foregoing experiments demonstrated the following: incorporation ofvancomycin into the silica-based matrix using the sol-gel technologyprovides a controlled drug release over time, starting upon immersion(and thus implantation) and continuing for at least 3 weeks; and theemployed room temperature, low acidity, low alcohol concentrationsol-gel procedure did not alter the vancomycin properties sincevancomycin released from the sol-gel derived material is as effective ininhibiting bacteria as vancomycin solutions that were not obtained froma sol-gel carrier.

EXAMPLE 5 Synthesis of Sol-Gel/Trypsin Inhibitor Composite

Sol-gel derived glass discs with trypsin-inhibitor incorporated insidetheir matrix have been successfully synthesized. Trypsin Inhibitor(SIGMA) is a protein with molecular weight of 21 kD. The sol-gel/proteincomposite contains 1-10 mg Trypsin Inhibitor (TI) per 150-200 mg disc.Protein elution was measurable in samples with 2 mg or greater ofprotein per disc.

The procedure used to synthesize 1 gram (by dry weight) of the sol-gelderived glass was as follows: 2.48 ml of TMOS (Aldrich, St. Louis, Mo.)was combined with 2 ml DI water and 0.68 ml of methanol in a 30 mlbeaker and mixed for 5 minutes using magnetic stirring. This resulted inan H₂ O/TMOS molar ratio of 10:1 and a methanol/TMOS molar ratio of 1:1.Then, 0.01 ml of 1N HCl was added to catalyze the sol-gel reaction. Thisresults in a clear one phase solution which is stirred for 15 minutes.The sol was cast in 0.8 ml volumes into polystyrene containers and thetrypsin inhibitor solution was added in a volume of 0.2 ml of 0.1Nacetic acid solution with protein concentration in the range of 1-10mg/ml. The solution was mixed with vortexing and the containers werecapped. Gelling occurred within 1-4 days after casting. The capped solwas allowed to gel and age for times ranging between 1 day to 2 weeksdepending on the desired porosity at room temperature. After aging, thesol-gel was allowed to dry at either room temperature or 37° C. byuncapping the casting container. Any liquid produced was decanted offthe solid. A higher drying temperature increased the porosity and rateof shrinkage. After drying, the resulting solid had lost 60-70% of itsoriginal weight due to evaporation of water and alcohol (methanol is aby-product of the sol-gel reactions).

The resulting solid material was a porous three-dimensionalnetwork/polymer of silica which releases incorporated biomolecules in acontrolled release fashion. The various processing parameters aredepicted in Table II. Sample designations are of the Formula SxxxCxxPxx(date cast), where "Sxxx" is the calculated % silica, "Cxx" is thecalculated % calcium oxide, and "Pxx" is the calculated % phosphorouspentoxide. The date cast is presented as the "last two digits of theyear.month.day." The trypsin inhibitor content and form of the sample isindicated in the basic Formula TI=X- sample shape! where "X" is theamount of trypsin inhibitor in mg per sample. The effect of pH ongelling time is apparent from Table II.

                                      TABLE II                                    __________________________________________________________________________    Processing Parameters                                                                     Units                                                             __________________________________________________________________________    Sample           S100 S100 S100 S100 S100                                     (Laboratory Designation)                                                                       (94.4.18)                                                                          (94.4.18)                                                                          (94.4.18)                                                                          (94.4.18)                                                                          (94.4.18)                                TI = amount of trypsin                                                                         TI = 2-                                                                            TI = 3-                                                                            TI = 5-                                                                            TI = 7.5-                                                                          TI = 10-                                 inhibitor in mg   granules!                                                                          granules!                                                                          granules!                                                                          granules!                                                                          granules!                               Water/TMOS Ratio 10   10   10   10   10                                       Methanol/TMOS Ratio                                                                            1    1    1    1    1                                        Acid Catalyst                                                                             ml and N                                                                           0.1 ml 1                                                                           0.1 ml 1                                                                           0.1 ml 1                                                                           0.1 ml 1                                                                           0.1 ml 1                                 (amount and concentration)                                                                     N HCl                                                                              N HCl                                                                              N HCl                                                                              N HCl                                                                              N HCl                                    % Silica calculated                                                                            100  100  100  100  100                                      % Calcium Oxide  0    0    0    0    0                                        calculated                                                                    % Phosphorous Pentoxide                                                                        0    0    0    0    0                                        calculated                                                                    pH as cast       2.3  2.4  2.8  3    3.2                                      Gelling Time                                                                              hours                                                                              168  144  120  72   24                                       Volume cast ml   1    1    1    1    1                                        Weight as cast                                                                            mg   1003.3                                                                             1009.2                                                                             1007 997.2                                                                              960.9                                    Total Protein in sample                                                                   mg   2    3    5    7.5  10                                       Aging Time  days 7    7    7    7    7                                        Drying Time days 5    5    5    5    5                                        Drying Temperature                                                                        C    20   20   20   20   20                                       (degrees centigrade)                                                          Weight after drying                                                                       mg   384.8                                                                              390.2                                                                              382.5                                                                              391.4                                                                              363                                      % Weight loss                                                                             %    61.6 61.3 62   60.8 62.1                                     __________________________________________________________________________

EXAMPLE 6 Trypsin Inhibitor Release

The initial release kinetic studies were carried out by immersing thesol-gel/protein composite (100 mg sol-gel/1 mg protein for each sample)in deionized water inside containers which were siliconized in order toreduce protein binding. The protein content in water was measured atdifferent time points. The water was replaced fresh after each timeperiod. The collected fluid that had been in contact with thesol-gel/protein composite was analyzed for protein content using acolloidal gold/spectrophotometric method (Integrated Separation Systems,Natick, Mass., Stoscheck et al., Anal. Biochem., (1987) 160:301-305,incorporated herein by reference) with sensitivity down to 0.5 μg/ml.The results are depicted in Table III below.

The numbers in the table represent protein release in μg of trypsininhibitor after immersion in DI water. Results for each time point areprovided together with cumulative protein release.

                  TABLE III                                                       ______________________________________                                                   Protein Released μg                                             Immersion Time:                                                                            3 days  1 week  2 weeks                                                                             3 weeks                                                                             4 weeks                              ______________________________________                                        Sample Designation*                                                           S100(94.4.18)TI2      75      38    20    16                                  cumulative release:   75     113   133   149                                  S100(94.4.18)TI3     115      56    33    27                                  cumulative release:  115     171   204   231                                  S100(94.4.18)TI5                                                                            75      82      48    30    33                                  cumulative release:                                                                         75     157     205   235   268                                  S100(94.4.18)TI7.5                                                                         142      97      70    68    78                                  cumulative release:                                                                        142     239     309   377   455                                  S100(94.4.18)TI10                                                                          175     125      80    55    67                                  cumulative release:                                                                        175     300     380   435   502                                  ______________________________________                                         *For key see Table I.                                                    

The protein release kinetics of the samples and results listed in TableII are depicted in FIG. 7b. Protein release was measured for a period offour weeks. "T12" (open squares) represents TI=2 from the table. "T13"(open circles) represents TI=3 from the table. "T15" (filled squares)represents TI=5 and T17.5 (filled circles) represents TI=7.5. "TI 10"(filled square within open square) represents TI=10 sample S100(94.4.18). All samples were in the form of granules having a diameterless than about 2 mm.

As can be seen from FIG. 7a, trypsin inhibitor was continually releasedfrom all samples for a period of at least seven (7) weeks.

EXAMPLE 7 Bioactivity of Pure Silica Glass

Sol-gel derived glass with a composition of 100% silica and water/TMOSmolar ratio of 15:1 was synthesized and its bioactivity tested in vitroin SPS by measuring changes in calcium-ion concentration. A 5 gramsample was made by combining 12.38 ml of TMOS with 8.87 ml of DI waterand sonicating for 5 minutes in an ice cooled bath. To this mixture,8.87 ml of 0.1N Acetic Acid was added and the mixture sonicated for anadditional 15 minutes. Then, 4.43 ml sodium phosphate (0.01M, pH 7)buffer was added and the mixture sonicated for one minute. The liquidsol was cast as 3 ml samples. The pH of the sol as cast was 4.5. Thegelling time was approximately 2 hours. Aging of the samples was done atroom temperature, for 1 day. Samples were dried for 3 days at 37° C. andweighed about 500 mg.

Samples in the form of discs approximately 1 cm in diameter and 4 mmhigh (1.76 cm² SA) were then immersed into SPS (17.6 ml) for a samplesurface area to immersion solution volume ratio of 0.1 cm⁻¹. Sampleswere immersed for two weeks with constant stirring at 37° C. SPSconcentration of calcium normally averages 100 ppm. After 2 weeks ofimmersion of samples, the average concentration of calcium in theretrieved SPS averaged 25 ppm. This indicates that calcium was consumedby the glass from solution, most likely by forming a calcium phosphatelayer on its surface.

EXAMPLE 8 Bioactivity of Silica-Based Glass Containing Other Oxides

Sol-gel samples with a composition of 65% SiO₂, 30% CaO and 5% P₂ O₅, byweight, were made by combining 1.61 ml TMOS, 5.04 ml 20% calciummethoxyethoxide solution in methoxyethanol, and 0.12 ml triethylphosphate, and magnetically stirring for 5 minutes at 4° C. To thismixture, 1 ml 1N HCl was added to mimick the conditions forincorporation of proteins, and stirred for an additional minute, for awater/TMOS molar ratio of 5.13. Four, 1 ml samples were cast and the solgelled in about 5 minutes. These samples were aged for 3 days, and thendried for 4 days at room temperature. Samples after drying weighed about600 mg.

Sol-gel samples were then immersed into 12 ml of SPS to test for calciumphosphate surface layer formation. Samples were retrieved after beingimmersed 5 days in SPS with constant stirring at 37° C. The samples wereviewed using scanning electron microscopy (SEM) and surface analysis wasperformed using energy dispersive x-ray analysis (EDXA). The surface ofthe samples contained nodules 1-3 μm in diameter (FIG. 1) that, whenanalyzed with EDXA (FIG. 2), contained high proportions of calcium andphosphorous. This indicates the formation of calcium phosphatenucleation sites as a precursor to calcium phosphate layer formation.

EXAMPLE 9 Synthesis of Sol-Gel/TGF-β Composite

Recombinant human transforming growth factor beta (TGF-β1) wasincorporated into pure silica sol-gel glass. The sol-gel was synthesizedby combining 5 ml TMOS with 5.4 ml of water, for a water/TMOS molarratio of 9:1, and magnetically stirring for 5 minutes at roomtemperature. 10 μL of 1N HCl was added to the mixture and the sol wasstirred for 30 minutes. Then, 0.9 ml of the sol was cast into apolystyrene container and 0.1 ml of TGF-β solution with 1% bovine serumalbumin (BSA) to prevent non-specific binding of the growth factor tothe casting container was added. Different quantities of TGF-β wereadded to each sample cast ranging from 0.5 μg to 2 μg of TGF-β for eachsolution added with 1% BSA. Samples were aged for 3 days at 37° C. anddried at 37° C. until they had lost approximately 50% of their "as cast"weight.

EXAMPLE 10 TGF-β1 Release

Sol-gel derived silica glass was synthesized by mixing and stirringTMOS, DI water, and 1N HCl in a molar ratio of 1:10:0.001. Then, 0.9 mlof the sol was cast into 15 ml diameter polystyrene vials and 0.1 ml ofa solution containing either 0.5 or 1 μg of TGF-β1 was added to the solsamples. TGF-β1 (Celtrix Lab., Inc.) was prepared according toinstructions supplied. Briefly, aliquots from a 2.347 μg/μl TGF-β1 stocksolution were resuspended in 10 mM HCl following lyophilization in 1%bovine serum albumin (BSA) (Sigma Chemical Co.). The resulting TGF-β1solutions were stored in 1.5 ml microcentrifuge tubes (U.S.A Scientific)at -70° C. until use. The pH of the sols at the time of casting wasmeasured to be 1.7. The vials were sealed and the sols were allowed togel (15 hours) and age (24 hours) in an incubator at 37° C. Then, thegels were dried to either 50 or 70% weight loss resulting in transparentglass disks about 10 mm in diameter. Part of the samples of each groupwas crushed to produce granules in a size range from about 500 to about1000 μm. A total of 4 sets of silica glass/TGF-β1 composites wereprepared as follows: 1 μg dose: disks and particles dried to 50% weightloss, and particles dried to 70% weight loss; 0.5 μg dose: particlesdried to 50% weight loss. An additional six silica glass diskscontaining 0.54 μg TGF-β1 and two controls (without TGF-β1) wereprepared and dried to 57% weight loss. The disks had uniform dimensionswith an average diameter of 10.17 mm and an average height of 4.93 mm.

The release of TGF-β1 from the sol-gel derived silica glass particlesand disks was measured by immersion in 1 ml of sterile phosphatebuffered saline (PBS) containing 1% BSA. Prior to immersion all thesamples were sterilized by UV irradiation. The BSA prevents thenon-specific binding of TGF-β1 to the immersion reservoir. Concentrationwas determined using an enzyme linked immunosorbent assay (ELISA).

The amount of active TGF-β1 released from the sol-gel materials wasassessed using the Mv1Lu mink lung epithelial cell inhibition assay.This assay determines TGF-β1 activity based on its inhibition of Mv1Lucell proliferation as measured by ³ H!-thymidine incorporation. Jenningset al., "Comparison of the biological activities of TGF beta 1 andTGF-beta 2: Differential activity in endothelial cells", J. CellPhysiol. 137:167-172 1988. Confluent Mv1Lu cells (ATCC) were lifted fromtissue culture flasks using Cell Dissociation Solution (Specialty Media)and plated in Corning 24-well polystyrene tissue culture dishes. Cellswere plated at a density 4.0×10⁴ cells/well in 1 ml of Dulbecco'sModified Eagle's Medium (DMEM) supplemented with 1% fetal bovine serum(FBS) (Hyclone) and 50 μg each of penicillin and streptomycin (SigmaCell Culture). Dishes were incubated at 37° C. and 5% CO₂ for 24 hoursto allow the cells to adhere to the bottom of the wells.

Following incubation, the wells were aspirated and treated with mediacontaining TGF-β1 of known picomolar (pM) concentrations as well aslyophilized 1% BSA in 10 mM HCl to serve as the control. Theconcentrations ranged from 0.1 pM to 10.0 pM and were added intriplicate. Aliquots from sample solutions, i.e. containing TGF-β1released from silica glass upon immersion, were diluted into the samerange of concentrations and also added to the cells. The dishes wereincubated for additional 24 hours.

After treatment with TGF-β1 standard and sample solutions, the wellswere aspirated and the cells labeled for 2 hours with 1 μCi/ml of ³H!-thymidine (NEN Research Products) in 1 ml of tissue culture medium.At the conclusion of the incubation period, the relative levels ofradioactivity incorporated into cellular DNA were assessed. Each wellwas washed with 1 ml of PBS, pH 7.4, followed by 10 minutes of treatmentwith trichloroacetic acid (TCA) to precipitate all unincorporated ³H!-thymidine. Following TCA precipitation, the cells were washed twicewith PBS and solubilized with 2% sodium dodecyl sulfate by shaking atroom temperature for 2 hours. Radioisotope incorporation into eachsample was determined by liquid scintillation counting of a 200 μlaliquots in 5 ml of ICN Ecolume scintillation fluid using a BeckmanLS1800 Liquid Scintillation Counter. Duplicate counts were performed foreach sample.

The effect of various parameters such as the concentration ofincorporated TGF-β1, the degree of drying, and the surface area tovolume ratio, on the cumulative release of biologically active TGF-β1vs. time is depicted in FIGS. 8, 9, and 10. A sustained release ofbiologically active TGF-β1 over a 7 day period, with maximum releaseoccurring at 3 days, was observed for the various group samples. Theamount of the released TGF-β1 depended on the processing parameters.Specifically, the released amount increased with the concentrationincorporated and decreased with the degree of drying. The release alsodepended on the material shape, i.e. SA/V ratio. With an increase in theincorporated content from 0.5 to 1 μg the amount released from granulesincreased from 3.4 to 10.5 ng after 7 days of immersion (FIG. 8). Thecumulative release also increased significantly with a reduction in adegree of drying from 70 to 50% (FIG. 9). Moreover, the release fromsmall granules was 3 times greater than that from disks due to asignificant increase in the SA/V ratio from 1.1 to 10 mm⁻¹ (FIG. 10).Thus, among the experimental groups the largest released amount of 10.5ng, equal to 1% of the incorporated amount, was observed in the case ofsamples loaded with 1.0 μg TGF-β1, dried to 50% weight loss, and crushedto granules in a size range from 500 to 1000 μm. The measurements,conducted in triplicate, confirmed a sustained release of biologicallyactive TGF-β1 over 7 days of immersion (FIG. 11).

The sol-gel technology used to synthesize glass/TGF-β1 composites doesnot alter the biological functionality of TGF-β1 The carriers showed asustained release of therapeutic quantities of the osteoinductive growthfactor in its biologically active form.

EXAMPLE 11 Synthesis and Characterization of Ca and P Containing Glasses

A sol-gel derived silica-based glass containing Ca and P was synthesizedby mixing the three alkoxides TMOS, CME, and TEP under an argonatmosphere and stirring the mixture for 5 minutes using a magneticstirrer. A glass having a final composition of about 70% SiO₂, 15% CaO,and 5% P₂ O₅ (percent dry weight) was prepared by mixing 3,47 ml TMOS,8.4 ml CME, and 0.24 ml TEP. Then, 1.1 ml of the sol was cast per 15 mmdiameter polystyrene vial and 0.38 ml of 0.1N acetic acid was added toeach of the sols to mimick the conditions for incorporation of proteins.The gels were sealed, aged for three days at room temperature, and driedat 37° C. to 50% weight loss.

The microstructure of the Ca-P containing silica-based glass preparedwas characterized using surface area analysis (Autosorb-1,Quantachrome). Prior to the analysis, the samples were outgassed at 30°C. The material pore structure can be characterized by the shape ofabsorption isotherms, i.e. plots representing changes in the absorbedgas (N₂) volume vs. relative pressure P/P₀. The isotherm for the Ca-Pcontaining silica-based glass is depicted in FIG. 12. The shape of theisotherm is characteristic of a mesoporous material, as defined in theManual on Using a Surface Analyzer Autosorb1, Quantachrome Corp., pp.II-4-46, 1992, incorporated herein by reference. Mesoporous materialsare defined therein as materials having an intermediate pore size, orpores in the size range of greater than 20 angstroms and less than 500angstroms. SSA, PV, and mean pore size were determined to be 331 m² /g,0.97 cc/g, and 58.4 angstroms, respectively.

The ability of the synthesized Ca-P containing glass to form a surfaceHA layer was assayed after immersion in SPS for one week. The sampleswere analyzed prior to and after immersion using FTIR. The FTIR spectraof the samples before and after one week of immersion are presented inFIG. 13. The absorption bands in the spectrum before immersion (bottomspectrum) are characteristic of silica-gel. After immersion, a doubletof bands appeared at 603 and 580 cm⁻¹ (upper spectrum). These bandsindicate the formation of HA on the glass surface, thereby indicatingbioactive behavior.

EXAMPLE 12 Synthesis of Ca and P containing Sol-Gel TI Composite

Sol-gel derived glass containing Si, Ca, and P was synthesized bycombining TMOS (3.47 ml), CME (8.40 ml) and TEP (0.24 ml), to produce acomposition of 70% SiO₂, 25% CaO and 5% P₂ O₅, under an argon atmosphereand mixing for 15 minutes. For each sample, 0.75 ml of the alkoxidemixture was cast in a polystyrene container and then mixed by vortexingwith 0.25 ml of 0.1N Acetic acid/protein solution (TI concentration=2,3, 4, 5 mg). The water/TMOS ratio was 10:1 and the gelling of all thesamples occurred in under 1 minute. Aging took place over 3 days insealed containers at room temperature. The samples were dried to 50% oftheir as-cast weight at 37° C. by uncapping their containers. Afterdrying, the samples were crushed to produce granules in a size range ofabout 100-1000 μm in diameter.

EXAMPLE 13 Trypsin Inhibitor Release

Protein release studies were performed by immersing 500 mg of thegranules in 1 ml of 50 mM tris buffer solution (pH 7.3 at 37° C.).Solutions were replaced completely after each time point varying from 1hour to seven weeks. Samples were immersed in 1 ml plain tris solutionat 37° C. with constant shaking. For each time point measured (1 hr, 2hrs, 4 hrs, 24 hrs, 48 hrs, 72 hrs, 96 hrs, 1 week, 2 wks, 3 wks, and 4wks) the solution was exchanged for fresh tris solution and the proteinconcentrations were measured using a colloidal gold assay. The proteinconcentrations in the solutions were measured using a gold colloidalassay (Integrated Separation Systems, Natick Mass.). Cumulative proteinrelease from the Ca-P containing glass is represented in FIG. 14.

A comparison with FIG. 7 reveals that a sustained release over longimmersion time periods was observed for both types of glasscompositions. In both cases, a somewhat rapid release was observed up toimmersion times between 4 and 7 days, after which a more gradual releasewas observed. The released amount depends on the TI concentrationincorporated, i.e. with a greater TI content in the glass matrix, theamount released was greater. A 10% release from the silica glass matrixwas measured after 6 weeks of immersion, while a 5% release from theCa-P containing glass was obtained after 4 weeks of immersion. Theaddition of Ca and P to the sol-gel derived silica glass did notsignificantly affect the TI release profile and the released amount.

EXAMPLE 14 Bioactivity Studies Including Sol-Gels with Trypsin Inhibitor

Samples were synthesized in a similar way as described in Example 12above, except that the proportion of alkoxides was changed to achievethree different compositions:

(1) 70% SiO₂, 25% CaO and 5% P₂ O₅

(2) 87% SiO₂, 10% CaO and 3% P₂ O₅

(3) 94% SiO₂, 5% CaO and 1% P₂ O₅

Composition (1) was synthesized both with and without 4 mg of TI, thetwo other compositions were synthesized without TI. The samples wereimmersed as 25 mg of granules into 25 ml of SPS solution (a trisbuffered solution with electrolyte concentrations similar to plasma) at37° C. with constant shaking. At the end of the immersion period (either1, 3, or 7 days) the SPS solution was pipetted out and then analyzed forCa and PO₄ using atomic absorption spectrometry and colorimetry. Thegranules after immersion were analyzed after immersion using FTIR forthe presence of P--O bend peaks at around 600 cm⁻¹.

The FTIR spectra of glass of composition (1) prior to and afterimmersion in SPS for one week are represented in FIG. 15. The spectrumof the sample prior to immersion (lower spectrum) shows absorption bandsof silica and proteins in the lower (below 1200 cm⁻¹) and higher (above1200 cm⁻¹) energy regions, respectively. A doublet of bands, located at562 and 603 cm⁻¹, appeared in the spectrum after immersion (upperspectrum). The doublet, characteristic of the P--O bending mode ofvibration, indicates formation of a hydroxyapatite (HA) layer on thesurface of sol-gel derived glass. Formation of the HA layer was alsodetected on the glass of composition (1) without TI. Compositions (2)and (3) also showed formation of the HA layer.

The foregoing examples are meant to illustrate the invention and not tolimit it in any way. Those skilled in the art will recognize thatmodifications can be made which are within the spirit and scope of theinvention as set forth in the appended claims.

All references cited herein are incorporated herein by reference.

What is claimed is:
 1. A material for filling bony defects comprising animplant and a coating on at least a portion of said implant, saidcoating comprising a controlled release carrier, said carrier comprisinga silica-based glass having a porous matrix and biologically activemolecules incorporated throughout said matrix wherein said carrier isprepared using a sol-gel derived process, and said biologically activemolecules are incorporated during the liquid phase of the sol, at atemperature of about 40° C. or below.
 2. The material of claim 1 whereinsaid implant consists of bioactive glass.
 3. The material of claim 2wherein said bioactive glass is in granular form.
 4. The material ofclaim 1 wherein said implant comprises a prosthetic device.